Grandt, Kristin2020-05-042020-05-042020-01https://hdl.handle.net/11299/213056University of Minnesota M.S. thesis. January 2020. Major: Plant and Microbial Biology. Advisor: Neil Olszewski. 1 computer file (PDF); 69 pages.SPINDLY (SPY) is an O-fucosyltransferase involved in several processes in Arabidopsis thaliana, including gibberellin signaling, cytokinin signaling, and plant development. spy plants display a number of easily scored phenotypes such as increased stem elongation, decreased leaf serration, and early flowering. Despite its apparent importance, SPY’s function has not been fully characterized, as very few SPY substrates have been identified. In order to address this gap in knowledge, a suppressor screen was conducted to identify potential SPY interactors. An M2 population of ethyl methanesulfonate mutagenized spy-4 plants was screened for suppression of spy phenotypes. Three alleles that showed suppression of spy early flowering and spindly floral shoot phenotypes were identified. The strongest of those alleles was selected for analysis. However, spy-4 is partially male sterile, so the suppressor allele was crossed into spy-3 to facilitate genotypic and phenotypic analysis. Bulk segregant analysis using next generation sequencing was employed to identify the mutation responsible for the partial spy suppression observed. This analysis identified a point mutation that introduces a premature stop codon into the coding region of the TRM5a gene as the most likely candidate. This EMS allele is called trm5a-1 here-in. Analysis of another trm5a allele (T-DNA insertion line trm5a-2) and transgene rescue experiments confirmed that this mutation was responsible for suppressing spy. trm5a-1 and trm5a-2 mutants grow slowly and flower late, phenotypes that have been previously reported in other trm5a alleles. The trm5a-1/spy-3 double mutant phenotypes for these traits suggest a complex interaction between the two genes. Other phenotypes which have not been previously characterized in trm5a mutants are also observed. Exogenous application of cytokinin results in outgrowths at the valve margin in trm5a-1, trm5a-2, spy-3, and trm5a-1/spy-3, but not, at the concentrations used, in Col-0. In all trm5a-1, trm5a-2, and spy-3 single mutants, the outgrowths are small hair- or fan-like protrusions . However, in trm5a-1/spy-3, the outgrowths are larger and may even appear to have stigmatic papillae on their surface. There is also a phenotype of floral clustering in trm5a mutants, which is occasionally observed in some spy mutants but not spy-3. In trm5a-1 and trm5a-2 mutants, the phenotype manifests mainly as flower doublets with occasional small clusters of 4-5 flowers, while in trm5a-1/spy-3 the clusters are larger often comprised of ten or more flowers. The trend of trm5a-1/spy-3 plants to display more severe valve margin outgrowth and clustering phenotypes than the single mutants suggests additive or synergistic interactions between TRM5a and SPY. Broadly, the phenotypes of the trm5a-1, trm5a-2, and spy-3 demonstrate the importance of the genes in proper plant growth and development, and the phenotypes observed in the trm5a-1/spy-3 double mutant suggest a degree of genetic interaction between the two genes and areas of interest for future research into both.enArabidopsisdevelopmentgrowthO-fucosyltransferasetRNA (guanine-N1-)-methyltransferaseThesis or Dissertation